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WO2021033990A1 - Composition comprenant des exosomes dérivés d'un précurseur de cellule souche mésenchymateuse dérivée d'une cellule souche pluripotente induite pour la prévention ou le traitement de la stéatohépatite non alcoolique - Google Patents

Composition comprenant des exosomes dérivés d'un précurseur de cellule souche mésenchymateuse dérivée d'une cellule souche pluripotente induite pour la prévention ou le traitement de la stéatohépatite non alcoolique Download PDF

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WO2021033990A1
WO2021033990A1 PCT/KR2020/010640 KR2020010640W WO2021033990A1 WO 2021033990 A1 WO2021033990 A1 WO 2021033990A1 KR 2020010640 W KR2020010640 W KR 2020010640W WO 2021033990 A1 WO2021033990 A1 WO 2021033990A1
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stem cells
mesenchymal stem
cells
derived
induced pluripotent
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Korean (ko)
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김수
이슬기
김지민
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Brexogen Inc
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Brexogen Inc
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Priority claimed from KR1020200097398A external-priority patent/KR102386464B1/ko
Application filed by Brexogen Inc filed Critical Brexogen Inc
Priority to US17/621,743 priority Critical patent/US20220233601A1/en
Priority to EP20855301.6A priority patent/EP4019027B1/fr
Priority to CN202080043654.6A priority patent/CN114025776B/zh
Priority to JP2022510957A priority patent/JP7534055B2/ja
Publication of WO2021033990A1 publication Critical patent/WO2021033990A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0662—Stem cells
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present invention is the prevention of non-alcoholic steatohepatitis comprising as an active ingredient exosomes derived from mesenchymal stem cells differentiated from induced pluripotent stem cell-derived mesenchymal stem cell progenitor cells with or without pretreatment It relates to a composition for treatment.
  • Mesenchymal stem cells are stromal cells with multipotency, and refer to cells that can differentiate into various cells including osteoblasts, chondrocytes, muscle cells, and adipocytes. Since mesenchymal stem cells can differentiate into various connective tissues such as cartilage, bone tissue, ligaments, and bone marrow matrix, studies are being conducted for various diseases such as treating soft tissue defects caused by arthritis, trauma, and burns.
  • non-alcoholic fatty liver is characterized by the accumulation of triglycerides, which are triglycerides, in liver cells without excessive alcohol consumption.
  • Non-alcoholic fatty liver continues to increase due to the overnutrition associated with high fat and high carbohydrate intake in modern people.
  • non-alcoholic fatty liver is commonly observed, but various factors are known to be related to non-alcoholic fatty liver. It is reported that 80% of adults with non-alcoholic fatty liver develop metabolic disorders such as insulin-resistant diabetes and heart disease.
  • Non-alcoholic fatty liver is classified into non-alcoholic simple steatosis and non-alcoholic steatohepatitis (NASH) with inflammation.If left untreated for a long time, hepatitis, liver fibrosis, cirrhosis, etc. Can lead to serious liver disease.
  • Non-alcoholic fatty liver is characterized by having an accumulation of fat (fat infiltration) in hepatocytes.
  • Non-alcoholic simple fatty liver can progress to non-alcoholic steatohepatitis.
  • fat accumulation is associated with varying degrees of inflammation and scarring of the liver, and in many cases associated with insulin resistance, dyslipidemia and hypertension. Is known.
  • Nonalcoholic steatohepatitis often occurs in people who are overweight, have high cholesterol and triglyceride levels, and/or insulin resistance.
  • non-alcoholic steatohepatitis In general, for the treatment of non-alcoholic steatohepatitis, obesity drugs, insulin resistance drugs, hyperlipidemia drugs, hepatocyte protective agents, antioxidants, etc. are used. However, these drugs are not essential treatments for non-alcoholic steatohepatitis, but are used as symptom-improving drugs and have side effects when taken for a long time.
  • the present inventors made intensive research efforts to develop a therapeutic agent for non-alcoholic steatohepatitis using exosomes of mesenchymal stem cells.
  • mesenchymal stem cells differentiated from progenitor cells of mesenchymal stem cells that are in the undifferentiated stage than mesenchymal stem cells derived from induced pluripotent stem cells (iPSCs) are established and pretreated with a pretreatment material or Or it was found that exosomes derived from mesenchymal stem cells differentiated from the induced pluripotent stem cell-derived mesenchymal stem cell progenitor cells that were not pretreated exhibit excellent preventive or therapeutic effects of non-alcoholic steatohepatitis, and the present invention Finished.
  • an object of the present invention is a non-alcoholic steatohepatitis comprising exosomes isolated from induced pluripotent stem cells (iPSC)-derived mesenchymal stem cells (MSC) as an active ingredient.
  • iPSC induced pluripotent stem cells
  • MSC mesenchymal stem cells
  • NASH -Alcoholic steatohepatitis
  • Another object of the present invention is to provide an exosome isolated from induced pluripotent stem cells-derived mesenchymal stem cell cells.
  • Another object of the present invention is a non-alcoholic steatohepatitis (Non-alcoholic) comprising exosomes isolated from mesenchymal stem cells (MSC) pretreated with a pretreatment material as an active ingredient.
  • steatohepatitis to provide a pharmaceutical composition for the prevention or treatment.
  • Another object of the present invention is to provide an exosome isolated from an induced pluripotent stem cell-derived mesenchymal stem cell pretreated with a pretreatment material.
  • the present inventors made intensive research efforts to develop a therapeutic agent for non-alcoholic steatohepatitis using exosomes of mesenchymal stem cells.
  • mesenchymal stem cells differentiated from progenitor cells of mesenchymal stem cells that are in the undifferentiated stage than mesenchymal stem cells derived from induced pluripotent stem cells (iPSCs) are established and pretreated with a pretreatment material or
  • iPSCs induced pluripotent stem cells
  • the present invention is an exosome isolated from mesenchymal stem cells differentiated from progenitor cells of induced pluripotent stem cells-derived mesenchymal stem cells, and a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis comprising the same as an active ingredient, and It relates to a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis comprising the exosomes isolated from the induced pluripotent stem cells-derived mesenchymal stem cells pretreated with a pretreatment material and the same as an active ingredient.
  • One aspect of the present invention is a non-alcoholic steatohepatitis comprising exosomes isolated from induced pluripotent stem cells (iPSC)-derived mesenchymal stem cells (MSCs) as an active ingredient (Non- alcoholic steatohepatitis) to prevent or treat pharmaceutical composition.
  • iPSC induced pluripotent stem cells
  • MSCs mesenchymal stem cells
  • Another aspect of the present invention relates to a pharmaceutical composition for preventing, alleviating, improving or treating non-alcoholic steatohepatitis comprising exosomes isolated from induced pluripotent stem cells-derived mesenchymal stem cells as an active ingredient.
  • non-alcoholic fatty liver disease in the present specification is one of nonalcoholic fatty liver disease (NAFLD) and is a progressive liver disease characterized by inflammation or fibrosis along with fatty liver, which causes cirrhosis or liver cancer. It means a precursor disease.
  • NAFLD nonalcoholic fatty liver disease
  • stem cell refers to a cell having the ability to differentiate into two or more different types of cells while having the ability to self-replicate as an undifferentiated cell.
  • the stem cells of the present invention may be autologous or allogeneic stem cells.
  • induced pluripotent stem cell refers to a cell that induces dedifferentiation in already differentiated cells, such as somatic cells, to return to an initial undifferentiated state and has pluripotency.
  • the dedifferentiation can be induced by introducing and expressing a specific gene (eg, Sox2, c-Myc, Klf4, Oct-4, etc.) or by injecting a dedifferentiation inducing protein made in a cell into which the specific gene has been introduced. .
  • a specific gene eg, Sox2, c-Myc, Klf4, Oct-4, etc.
  • the pluripotency refers to the ability to differentiate into tissues or organs of three germ layers constituting a living body, namely endoderm, mesoderm and ectoderm.
  • mesenchymal stem cell refers to a stem cell capable of differentiating into various cells including osteoblasts, chondrocytes, muscle cells, adipocytes, etc. as cells having multipotency.
  • the mesenchymal stem cells are most commonly used as bone marrow-derived mesenchymal stem cells, but may be derived from umbilical cord or umbilical cord blood, adipose tissue, amniotic fluid, tooth buds of molars in addition to bone marrow.
  • Mesenchymal stem cells are also called stromal cells.
  • the progenitor cells of the mesenchymal stem cells are not general mesenchymal stem cells, but are progenitor cells of mesenchymal stem cells derived from induced pluripotent stem cells (iPSCs) [developed by the present inventors].
  • iPSCs induced pluripotent stem cells
  • the progenitor cells of mesenchymal stem cells derived from induced pluripotent stem cells may not express a stage-specific embryonic antigen 4 (SSEA-4) protein.
  • SSEA-4 stage-specific embryonic antigen 4
  • the mesenchymal stem cells derived from iPS cells may be differentiated from progenitor cells of mesenchymal stem cells derived from iPS cells that do not express a stage-specific embryonic antigen 4 (SSEA-4) protein.
  • SSEA-4 stage-specific embryonic antigen 4
  • the "derived pluripotent stem cell” refers to cells induced to have pluripotent differentiation ability through an artificial dedifferentiation process from differentiated cells, and is also referred to as a dedifferentiated stem cell.
  • the artificial dedifferentiation process is performed by introduction of a non-viral-mediated dedifferentiation factor using a virus-mediated or non-viral vector using retrovirus, lentivirus, and Sendai virus, protein and cell extract, or stem cell extract. , Dedifferentiation by compounds, etc.
  • the induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, specifically show similar cell shapes, similar gene and protein expression patterns, have pluripotency in vitro and in vivo, and teratoma When inserted into a blastocyst of a mouse, chimera mice are formed, and germline transmission of genes is possible.
  • the induced pluripotent stem cells of the present invention include induced pluripotent stem cells derived from all mammals such as humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, rabbits, but preferably human induced pluripotent stem cells. It is a cell.
  • somatic cells before the induced pluripotent stem cells of the present invention are dedifferentiated may be somatic cells derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, amniotic fluid, or placenta, but is not limited thereto.
  • the somatic cells are fibroblasts, hepatocytes, adipocytes, epithelial cells, epidermal cells, chondrocytes, muscle cells, and myocardial cells. Includes, but is limited to, cardiac muscle cells, melanocytes, neural cells, glial cells, astroglial cells, monocytes, macrophages, etc. It does not become.
  • the mesenchymal stem cells of the present invention are ANKRD1, CPE, NKAIN4, LCP1, CCDC3, MAMDC2, CLSTN2, SFTA1P, EPB41L3, PDE1C, EMILIN2, compared to an equal number of other mesenchymal stem cells.
  • One or more genes selected from the group consisting of SULT1C4, TRIM58, DENND2A, CADM4, AIF1L, NTM, SHISA2, RASSF4, and ACKR3 are expressed at higher levels.
  • the mesenchymal stem cells of the present invention are at least one gene selected from the group consisting of DHRS3, BMPER, IFI6, PRSS12, RDH10, and KCNE4 compared to the same number of other mesenchymal stem cells. Is expressed at a lower level.
  • the mesenchymal stem cells and an equal number of other mesenchymal stem cells are derived from allogeneic tissue. More specifically, the mesenchymal stem cells are progenitor cells of mesenchymal stem cells derived from induced pluripotent stem cells.
  • the mesenchymal stem cells are mesenchymal stem cells derived from induced pluripotent stem cells derived from umbilical cord tissue, and the equivalent number of other mesenchymal stem cells compared thereto are mesenchymal stem cells derived from umbilical cord tissue. It is a stem cell.
  • the present inventors named mesenchymal stem cells differentiated from progenitor cells of mesenchymal stem cells derived from the induced pluripotent stem cells as BxC (brexogen stem cells).
  • BxC induced pluripotent stem cell-derived mesenchymal stem cell
  • BxC induced pluripotent stem cell-derived mesenchymal stem cell
  • progenitor cells of mesenchymal stem cells derived from induced pluripotent stem cells refers to cells at the stage just before being completely differentiated from induced pluripotent stem cells into mesenchymal stem cells, and is a kind of mesenchymal stem cells derived from induced pluripotent stem cells. It can be seen as, and refers to a cell that does not express the SSEA-4 protein and has the properties of a complete mesenchymal stem cell through additional culture.
  • BxC Mesenchymal stem cells of induced pluripotent stem cells derived from the same tissue (eg umbilical cord tissue) of the present invention have an abnormality in chromosomal karyotype compared to mesenchymal stem cells (MSC) derived from the same tissue (eg umbilical cord tissue). No, it has excellent proliferation ability. Specifically, the BxC of the present invention exhibits a difference in proliferative capacity of at least 10 times compared to that of mesenchymal stem cells (MSC) derived from the same tissue when passages are repeated 9 or more times. No reduction was observed. In addition, the expression level of Ki67, a marker related to cell proliferation ability, is more than twice as high in BxC as compared to MSC.
  • the induced pluripotent stem cell-derived mesenchymal stem cells contain an equal number of functional proteins such as Endostatin, Endothelin-1, VEGF-A, Thrombospondin-2, PlGF, PDGF-AA, beta-NGF, and HB-EGF. Compared to mesenchymal stem cells, it secretes a large amount.
  • Endostatin is a 20 kDa C-terminal fragment derived from naturally produced type XVIII collagen, and is reported as an anti-angiogenic agent.
  • endothelin-1 also known as preproendothelin-1 (PPET1)
  • PPET1 preproendothelin-1
  • EDN1 vascular endothelial cells
  • endothelin-1 is known as a powerful vasoconstrictor.
  • vascular endothelial growth factor A is a protein encoded by the VEGFA gene and is known to induce blood vessel growth through interaction with VEGFR1 and VEGFR2 of vascular endothelial cells.
  • Thrombospondin-2 is a protein encoded by the THBS2 gene, and is known to mediate cell-cell interaction or cell-substrate interaction. Although the role of Thrombospondin-2 on cancer is controversial, it has been reported to regulate cell surface properties of mesenchymal stem cells and is known to be involved in cell adhesion and cell migration.
  • the PLGF placental growth factor
  • the PLGF is a protein encoded by the PGF gene and is known as a protein that plays a major role in angiogenesis in the embryogenesis as a member of the VEGF sub-family.
  • the PDGF-AA platelet-derived growth factor
  • the PDGF-AA is a growth factor that regulates cell growth and division, and plays an important role in the generation and growth of blood vessels, and proliferation, chemotaxis, and migration of mesenchymal stem cells. It is known to do.
  • NGF Neuro growth factor
  • gamma-NGF acts as a serine protease
  • beta-NGF the N-terminal of beta-NGF It is known to activate NGF by cutting.
  • HB-EGF heparin-binding EGF-like growth factor
  • HBEGF human epidermal growth factor
  • exosome refers to a membrane vesicle having a membrane structure composed of a lipid-bilayer existing in cells or secreted by a cell extracellularly, and is present in the body fluids of almost all eukaryotes.
  • the diameter of exosomes is about 30-1000 nm, and when multivesicular bodies fuse with the cell membrane, they are released from the cell, or are released immediately from the cell membrane.
  • exosomes play a role in transporting proteins, bioactive lipids and RNA (miRNA), which are biomolecules within cells, in order to play a functional role in mediating coagulation, cell-cell communication and cellular immunity.
  • the exosome is a concept encompassing microvesicles.
  • marker proteins for exosomes CD63 and CD81 are known.Other than that, receptors on the cell surface such as EGFR, molecules related to signal transduction, cell adhesion related proteins, MSC related antigens, heat shock proteins, and vesicle formation Proteins such as Alix are known.
  • the exosomes isolated from the induced pluripotent stem cells-derived mesenchymal stem cells are present in the above-described induced pluripotent stem cells-derived mesenchymal stem cells (BxC), or they mean exosomes secreted from BxC.
  • exosomes isolated from induced pluripotent stem cells-derived mesenchymal stem cells differentiate from progenitor cells of mesenchymal stem cells derived from induced pluripotent stem cells that do not express the stage-specific embryonic antigen 4 (SSEA-4) protein. It may be isolated from the mesenchymal stem cells.
  • SSEA-4 stage-specific embryonic antigen 4
  • exosomes isolated from induced pluripotent stem cells-derived mesenchymal stem cells contain an amount sufficient to achieve prophylactic or therapeutic activity of non-alcoholic steatohepatitis. do.
  • the pharmaceutical composition comprises exosomes isolated from induced pluripotent stem cells-derived mesenchymal stem cells based on exosome protein 1 to 10000 ⁇ g, 1 to 1000 ⁇ g, 10 to 10000 ⁇ g, 10 to 1000 ⁇ g, 100 to 10000 ⁇ g, 100 to 1000 ⁇ g, 50 to 10000 ⁇ g, 50 to 1000 ⁇ g, or 50 to 500 ⁇ g may be included, but is not limited thereto.
  • prevention refers to all actions of inhibiting or delaying the progression of non-alcoholic steatohepatitis by administration of the composition of the present invention.
  • treatment refers to (a) inhibition of the development of non-alcoholic steatohepatitis; (b) alleviation of non-alcoholic steatohepatitis; And (c) it means the removal of non-alcoholic steatohepatitis.
  • the pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient.
  • the pharmaceutically acceptable carrier is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, but are not limited thereto.
  • a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like may be additionally included in addition to the above components.
  • composition of the present invention can be administered orally or parenterally (for example, intravenous, subcutaneous, intraperitoneal or topically applied) according to a desired method, and the dosage is It depends on the degree, drug form, administration route and time, but may be appropriately selected by those skilled in the art.
  • composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective amount is the type, severity, activity of the drug, and It can be determined according to sensitivity, administration time, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.
  • the pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.
  • the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient in the body, inactivation rate and excretion rate, the type of disease, and the drug used in combination.
  • Another aspect of the present invention relates to an exosome isolated from induced pluripotent stem cells-derived mesenchymal stem cells.
  • the exosomes (BxC-e) isolated from mesenchymal stem cells differentiated from progenitor cells of the induced pluripotent stem cells-derived mesenchymal stem cells of the present invention are exosomes having the characteristics of conventional exosomes.
  • the exosomes isolated from the progenitor cells of the induced pluripotent stem cells-derived mesenchymal stem cells had excellent inhibitory effects on adipogenic differentiation (FIG. 2).
  • the BxC-e of the present invention inhibits adipogenesis, inhibits inflammation, and suppresses endoplasmic reticulum stress in stem cells induced steatosis (FIGS. 5 to 7 ).
  • Another aspect of the present invention relates to a method for treating nonalcoholic steatohepatitis comprising administering to an individual an exosome isolated from the induced pluripotent stem cell-derived mesenchymal stem cells.
  • the "individual” refers to a subject in need of treatment of a disease, and more specifically, refers to a mammal such as a human or non-human primate, mouse, dog, cat, horse, and cow.
  • Another aspect of the present invention relates to the use of exosomes isolated from the induced pluripotent stem cells-derived mesenchymal stem cells for the treatment of non-alcoholic steatohepatitis.
  • the non-alcoholic steatohepatitis treatment method and therapeutic use are the exosomes isolated from the induced pluripotent stem cell-derived mesenchymal stem cells of the present invention described above, and the pharmaceutical composition and components including the same, so that Description of the common content is omitted in order to avoid excessive complexity of the present specification.
  • Another aspect of the present invention is a pre-treated with a pre-treatment material, induced pluripotent stem cells (iPSC)-derived mesenchymal stem cells (mesenchymal stem cells, MSC) containing the isolated exosomes as an active ingredient. It relates to a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis.
  • iPSC induced pluripotent stem cells
  • MSC mesenchymal stem cells
  • the mesenchymal stem cells derived from iPS cells may be differentiated from progenitor cells of mesenchymal stem cells derived from iPS cells that do not express a stage-specific embryonic antigen 4 (SSEA-4) protein.
  • SSEA-4 stage-specific embryonic antigen 4
  • pretreatment refers to contacting the cell culture medium to which the pretreatment material is added to the progenitor cells of the induced pluripotent stem cell-derived mesenchymal stem cell Means process.
  • the pretreatment may be performed by culturing the induced pluripotent stem cell-derived mesenchymal stem cells in a cell culture medium containing a pretreatment material.
  • the cell culture medium can be any medium commonly used for animal cell culture, for example, DMEM (Dulbecco's modification of Eagle's medium), a mixture of DMEM and F12, Eagles' MEM (Eagle's minimum essential medium), ⁇ -MEM , Iscove's MEM, 199 medium, CMRL 1066, RPMI 1640, F12, F10, Way-mouth's MB752/1, McCoy's 5A and MCDB series, and the like can be used.
  • DMEM Dulbecco's modification of Eagle's medium
  • Eagles' MEM Eagles' MEM (Eagle's minimum essential medium)
  • ⁇ -MEM Iscove's MEM
  • 199 medium e.g., CMRL 1066, RPMI 1640, F12, F10, Way-mouth's MB752/1, McCoy's 5A and MCDB series, and the like
  • CMRL 1066 e.g., RPMI
  • the cultivation may be performed for 6 to 48 hours.
  • the culture is 6 to 42 hours, 6 to 36 hours, 6 to 30 hours, 6 to 27 hours, 12 to 48 hours, 12 to 42 hours, 12 to 36 hours, 12 to 30 hours, 12 to 27 hours , 18 to 48 hours, 18 to 42 hours, 18 to 36 hours, 18 to 30 hours, 18 to 27 hours, 21 to 48 hours, 21 to 42 hours, 21 to 36 hours, 21 to 30 hours or 21 to 27 hours Can be done.
  • the pretreatment material is 1-(benzothiazolylsulfonyl)-5-chloro-1 hydrogen-indole-2butanoic acid [1-(6-benzothiazolylsulfonyl)-5-chloro-1H-indole-2-butanoic acid] or exene It may be Exendin-4.
  • the 1-(benzothiazolylsulfonyl)-5-chloro-1 hydrogen-indole-2butanoic acid [1-(6-benzothiazolylsulfonyl)-5-chloro-1H-indole-2-butanoic acid] is called "Lanifibranor” It can be used as a name and is an agonist of peroxisome proliferator-activated receptors (PPARs).
  • PPARs peroxisome proliferator-activated receptors
  • the Lanifibranor may be included in a concentration of 1 to 100 ⁇ M in the cell culture medium.
  • the Lanifibranor is 1 to 90 ⁇ M, 1 to 80 ⁇ M, 1 to 70 ⁇ M, 1 to 60 ⁇ M, 1 to 50 ⁇ M, 1 to 40 ⁇ M, 1 to 30 ⁇ M, 10 to 90 ⁇ M in cell culture medium, 10 to 80 ⁇ M, 10 to 70 ⁇ M, 10 to 60 ⁇ M, 10 to 50 ⁇ M, 10 to 40 ⁇ M, and 10 to 30 ⁇ M.
  • Lanifibranor is 1 to 1000 ⁇ M, 1 to 500 ⁇ M, 1 to 100 ⁇ M, 1 to 90 ⁇ M, 1 to 80 ⁇ M, 1 to 70 ⁇ M, 1 to 60 ⁇ M, 1 to 50 ⁇ M, 1 to 40 ⁇ M, 1 to It may be added at a concentration of 30 ⁇ M, 1 to 20 ⁇ M, or 10 ⁇ M, but is not limited thereto.
  • the exosome is 1-(benzocyazolylsulfonyl)-5-chloro-1 hydrogen-indole-2butanoic acid is 1000 ⁇ M, 1 to 500 ⁇ M, 1 to 100 ⁇ M, 1 to 90 ⁇ M, 1 to 80 ⁇ M , 1 to 70 ⁇ M, 1 to 60 ⁇ M, 1 to 50 ⁇ M, 1 to 40 ⁇ M, 1 to 30 ⁇ M, 1 to 20 ⁇ M, 10 to 90 ⁇ M, 10 to 80 ⁇ M, 10 to 70 ⁇ M, 10 to 60 ⁇ M, 10 to 50 ⁇ M, It may be an exosome isolated from progenitor cells of induced pluripotent stem cells-derived mesenchymal stem cells cultured in a medium added at a concentration of 10 to 40 ⁇ M and 10 to 30 ⁇ M or 10 ⁇ M.
  • Exendin-4 of the present invention is a peptide agonist of a glucagon-like peptide (GLP) receptor. Exendin-4 is known to promote insulin secretion, and has been used clinically for treatment of type 2 diabetes and Parkinson's disease.
  • GLP glucagon-like peptide
  • the exendin-4 may be included in a concentration of 1 to 100 nM in the cell culture medium.
  • the exendin-4 is 1 to 90 nM, 1 to 80 nM, 1 to 70 nM, 1 to 60 nM, 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 10 to in the cell culture medium It may be included in a concentration of 90 nM, 10 to 80 nM, 10 to 70 nM, 10 to 60 nM, 10 to 50 nM, 10 to 40 nM, 10 to 30 nM or 20 nM.
  • the exosome is Exendin-4 is 1 to 90 nM, 1 to 80 nM, 1 to 70 nM, 1 to 60 nM, 1 to 50 nM, 1 to 40 nM, 1 to 30 nM , 10 to 90 nM, 10 to 80 nM, 10 to 70 nM, 10 to 60 nM, 10 to 50 nM, 10 to 40 nM, 10 to 30 nM, or induced pluripotent stem cells cultured in a medium added at a concentration of 20 nM It may be isolated from the progenitor cells of the derived mesenchymal stem cells, but is not limited thereto.
  • the pharmaceutical composition is an exo isolated from progenitor cells of induced pluripotent stem cells-derived mesenchymal stem cells pretreated with 1-(benzociazolylsulfonyl)-5-chloro-1 hydrogen-indole-2butanoic acid.
  • Exosomal protein based on 1 to 10000 ⁇ g, 1 to 1000 ⁇ g, 10 to 10000 ⁇ g, 10 to 1000 ⁇ g, 100 to 10000 ⁇ g, 100 to 1000 ⁇ g, 50 to 10000 ⁇ g, 50 to 1000 ⁇ g or 50 to 500 ⁇ g It may be included, but is not limited thereto.
  • the pharmaceutical composition comprises exosomes isolated from progenitor cells of induced pluripotent stem cells-derived mesenchymal stem cells pretreated with exendin-4, based on exosome protein 1 to 10000 ⁇ g, 1 to 1000 ⁇ g, 10 to 10000 ⁇ g, 10 to 1000 ⁇ g, 100 to 10000 ⁇ g, 100 to 1000 ⁇ g, 50 to 10000 ⁇ g, 50 to 1000 ⁇ g, or 50 to 500 ⁇ g may be included, but is not limited thereto.
  • Another aspect of the present invention relates to exosomes isolated from induced pluripotent stem cells-derived mesenchymal stem cells pretreated with a pretreatment material.
  • exosomes isolated from induced pluripotent stem cells-derived mesenchymal stem cells pretreated with the pretreatment material of the present invention are exosomes having characteristics as conventional exosomes.
  • BxC-V37e and BxC-G63e of the present invention inhibits adipogenesis, inhibits inflammation, and inhibits endoplasmic reticulum stress in hepatocytes induced by steatosis. It was confirmed (FIGS. 5 to 10).
  • Another aspect of the present invention is a food composition for alleviating, inhibiting, or improving non-alcoholic steatohepatitis comprising exosomes isolated from induced pluripotent stem cells-derived mesenchymal stem cells.
  • Another aspect of the present invention is a food composition for alleviation, inhibition, or improvement of non-alcoholic steatohepatitis comprising exosomes isolated from induced pluripotent stem cells-derived mesenchymal stem cells, pretreated with a pretreatment material.
  • the food composition according to the present invention has the exosomes isolated from the induced pluripotent stem cell-derived mesenchymal stem cells of the present invention described above, and the pharmaceutical composition and components including the same, the common contents between them The description is omitted to avoid excessive complexity of the specification.
  • the food composition according to the present invention may include ingredients that are commonly added during food production, and may include, for example, proteins, carbohydrates, fats, nutrients, seasoning agents, and flavoring agents, but is not limited thereto. .
  • Carbohydrates that can be included in the food composition according to the present invention include monosaccharides such as glucose fructose, disaccharides such as maltose, sucrose, and oligosaccharides, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol, and erythritol.
  • Sugar alcohols such as may be included, but are not limited thereto.
  • Flavoring agents that may be included in the food composition according to the present invention may include natural flavoring agents such as taumatin and stevia extract, and synthetic flavoring agents such as saccharin and aspartame, but are not limited thereto.
  • Another aspect of the present invention relates to a method for treating non-alcoholic steatohepatitis comprising administering to an individual exosomes isolated from induced pluripotent stem cells-derived mesenchymal stem cells pretreated with the pretreatment material.
  • the "individual” refers to a subject in need of treatment of a disease, and more specifically, refers to a mammal such as a human or non-human primate, mouse, dog, cat, horse, and cow.
  • Another aspect of the present invention relates to the use of exosomes isolated from induced pluripotent stem cells-derived mesenchymal stem cells pretreated with the pretreatment material for the treatment of non-alcoholic steatohepatitis.
  • the non-alcoholic steatohepatitis treatment method and treatment use are pre-treated with the pretreatment material of the present invention, the exosomes isolated from the induced pluripotent stem cells-derived mesenchymal stem cells, and a pharmaceutical composition and components including the same. Because of this, description of the common content between them is omitted in order to avoid excessive complexity of the present specification.
  • the present invention relates to a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis, comprising as an active ingredient exosomes isolated from induced pluripotent stem cells-derived mesenchymal stem cells that are pretreated or not pretreated with a pretreatment material.
  • the exosome of the present invention exhibits a more improved non-alcoholic steatohepatitis prevention or treatment effect compared to exosomes isolated from existing mesenchymal stem cells, and can be usefully used for related research and development and commercialization.
  • FIG. 1A is a diagram showing the average size and distribution of exosomes (BxC-e) isolated from induced pluripotent stem cells-derived mesenchymal stem cells (BxC).
  • 1B is an electron micrograph of exosomes (BxC-e) isolated from induced pluripotent stem cells-derived mesenchymal stem cells (BxC).
  • 2A is a photograph showing the effect of inhibiting lipodifferentiation of exosomes (BxC-e) isolated from induced pluripotent stem cells-derived mesenchymal stem cells (BxC).
  • 2B is a diagram showing the effect of inhibiting lipodifferentiation of exosomes (BxC-e) isolated from induced pluripotent stem cells-derived mesenchymal stem cells (BxC).
  • Figure 3a is a diagram showing the average size and distribution of exosomes (BxC-V37e) isolated from induced pluripotent stem cells-derived mesenchymal stem cells (BxC) of LANIFIBRANOR pretreatment.
  • Figure 3b is an electron micrograph of exosomes (BxC-V37e) isolated from induced pluripotent stem cells-derived mesenchymal stem cells (BxC) of LANIFIBRANOR pretreatment.
  • Figure 4a is a diagram showing the average size and distribution of exosomes (BxC-G63e) isolated from induced pluripotent stem cells-derived mesenchymal stem cells (BxC) of Exendin-4 pretreatment.
  • Figure 4b is an electron micrograph of exosomes (BxC-G63e) isolated from induced pluripotent stem cells-derived mesenchymal stem cells (BxC) of Exendin-4 pretreatment.
  • BxC-e is an exosome isolated from induced pluripotent stem cells-derived mesenchymal stem cells (BxC) in steatosis-induced stem cells and induced pluripotent stem cells-derived mesenchymal stem cells of pretreatment with LANIFIBRANOR ( BxC) isolated from exosomes (BxC-V37e) is a diagram showing the inhibitory effect of lipoogenesis (Lipogenesis).
  • FIGS. 6A and 6B show exosomes (BxC-e) isolated from induced pluripotent stem cells-derived mesenchymal stem cells (BxC) in steatosis-induced stem cells and induced pluripotent stem cells-derived mesenchymal stems of pretreatment with LANIFIBRANOR It is a diagram showing the effect of inhibiting inflammation (Inflammation) by exosomes (BxC-V37e) isolated from cells (BxC).
  • BxC-e is an exosome isolated from an induced pluripotent stem cell-derived mesenchymal stem cell (BxC) in steatosis-induced hepatocytes and an induced pluripotent stem cell-derived mesenchymal stem cell of pretreatment with LANIFIBRANOR ( BxC) is a diagram showing the effect of inhibiting endoplasmic reticulum stress (ER Stress) by exosomes (BxC-V37e) isolated from.
  • Figures 8a to 8c show the exosomes (BxC-G63e) isolated from the induced pluripotent stem cells-derived mesenchymal stem cells (BxC) of Exendin-4 and Exendin-4 pretreatment in steatosis-induced hepatocytes.
  • (Lipogenesis) is a diagram showing the inhibitory effect.
  • 9A and 9B show inflammation caused by exosomes (BxC-G63e) isolated from induced pluripotent stem cells-derived mesenchymal stem cells (BxC) of Exendin-4 and Exendin-4 pretreatment in steatosis-induced hepatocytes ( Inflammation) is a diagram showing the inhibitory effect.
  • Figure 10 shows endoplasmic reticulum stress (ER) isolated from exosomes (BxC-G63e) isolated from induced pluripotent stem cells-derived mesenchymal stem cells (BxC) of Exendin-4 and Exendin-4 pretreatment in steatosis-induced hepatocytes (ER It is a diagram showing the effect of suppressing stress).
  • Figure 11a is a normal group (Normal) that did not treat the substance in the liver cells of steatosis-induced mice, a control group treated with PBS (MCD-PBS), and a group treated with BxC-V37e (MCD-V37e) treated with BxC-V37e.
  • MCD-PBS PBS
  • MCD-V37e BxC-V37e
  • Figure 11b is a normal group (Normal) that did not treat the substance in the liver cells of steatosis-induced mice, the control group treated with PBS (MCD-PBS), and the BxC-V37e treated group (MCD-V37e) treated with BxC-V37e. This is a picture showing oil red O dyeing results.
  • Figure 12a is a normal group (Normal) that did not treat the substance in the liver cells of steatosis-induced mice, the control group treated with PBS (MCD-PBS), and the BxC-G63e treated group (MCD-G63e) treated with BxC-G63e.
  • MCD-PBS the control group treated with PBS
  • MCD-G63e the BxC-G63e treated group
  • H&E hematoxylin & eosin
  • Figure 12b is a normal group (Normal) that did not treat the substance in the liver cells of steatosis-induced mice, the control group treated with PBS (MCD-PBS), and the group treated with BxC-G63e treated with BxC-G63e (MCD-G63e). This is a picture showing oil red O dyeing results.
  • Figure 13a is a normal group (Normal) that did not treat the substance in the liver cells of steatosis-induced mice, the control group treated with PBS (MCD-PBS), and the BxC-V37e (MCD-V37e) treated group treated with BxC-V37e.
  • MCD-PBS PBS
  • MCD-V37e BxC-V37e
  • 13B is a normal group of mice treated with steatosis-induced hepatocytes without material treatment (Normal), a control group treated with PBS (MCD-PBS), and a group treated with BxC-G63e (MCD-V63e) treated with BxC-G63e.
  • Normal normal
  • MCD-PBS steatosis-induced hepatocytes without material treatment
  • MCD-V63e BxC-G63e
  • 14A is a view of the normal group (Normal), the control group treated with PBS (MCD-PBS), and the BxC-V37e treatment group (MCD-V37e) treated with a substance treated with steatosis-induced mice. This is a picture showing the results of immunohistochemical staining.
  • 14B is a view of the normal group (Normal), the control group treated with PBS (MCD-PBS), and the BxC-G63e treatment group (MCD-G63e) treated with a substance treated with steatosis-induced mice. This is a picture showing the results of immunohistochemical staining.
  • Figure 15a is a normal group (Normal) in which the material was not treated with the hepatocytes of mice in which fibrosis was induced, the control group treated with PBS (TAA-PBS), and the BxC-V37e (TAA-V37e) treated group treated with BxC-V37e. This is a picture showing liver tissue.
  • Figure 15b is a normal group (Normal) that was not treated with material to the hepatocytes of mice in which fibrosis was induced, a control group treated with PBS (TAA-PBS), and a group treated with BxC-G63e (TAA-G63e) treated with BxC-G63e.
  • This is a picture showing liver tissue.
  • Figure 16a is a normal group (Normal) that was not treated with material in the hepatocytes of mice in which fibrosis was induced, a control group treated with PBS (TAA-PBS), and a group treated with BxC-V37e (TAA-V37e) treated with BxC-V37e.
  • This is a picture showing the results of hematoxylin & eosin staining.
  • Figure 16b is a normal group (Normal) in which the material was not treated with the hepatocytes of mice in which fibrosis was induced, the control group treated with PBS (TAA-PBS), and the BxC-G63e (TAA-G63e) treated group treated with BxC-G63e.
  • This is a picture showing the results of hematoxylin & eosin staining.
  • Figure 17a is a normal group (Normal) in which the material was not treated with fibrosis-induced mouse hepatocytes, the control group treated with PBS (TAA-PBS), and the BxC-V37e (TAA-V37e) treated group treated with BxC-V37e. This is a photograph showing the results of picrosirius red staining.
  • Figure 17b is a normal group (Normal) that was not treated with material to the hepatocytes of mice in which fibrosis was induced, a control group treated with PBS (TAA-PBS), and a group treated with BxC-G63e (TAA-G63e) treated with BxC-G63e.
  • This is a photograph showing the results of picrosirius red staining.
  • iPSC induced pluripotent stem cells
  • MSC mesenchymal stem cells
  • % used to indicate the concentration of a specific substance is (weight/weight)% for solids/solids, (weight/volume)% for solids/liquids, and Liquid/liquid is (vol/vol)%.
  • iPSC induced pluripotent stem cells
  • BxC derived mesenchymal stem cells
  • induced pluripotent stem cells were cultured for 7 days in DMEM to which 10% FBS and 10 ng/ml bFGF were added.
  • SSEA-4 (-) cells that do not express SSEA-4 (stage-specific embryonic antigen 4) protein on the cell surface through FACS from cultured induced pluripotent stem cells induced pluripotent stem cells-derived mesenchyme Stem cell progenitor cells were obtained.
  • the isolated SSEA-4 (-) cells were passaged and further cultured for 7 days in the same medium as described above to prepare the induced pluripotent stem cells-derived mesenchymal stem cells of the present invention.
  • the present inventors named the induced pluripotent stem cell-derived mesenchymal stem cell as BxC (brexogen stem cell).
  • BxC Mesenchymal stem cells derived from induced pluripotent stem cells named BxC were cultured in culture medium [high glucose DMEM (Gibco, Cat no.11995-065), 10% Fetal bovine Serum (HyClone), 1% MEM Non-Essential Amino Acids Solution ( 100X) (Gibco, Cat no. 11140-050)].
  • Example 1 Isolation of induced pluripotent stem cells-derived mesenchymal stem cells (BxC)-derived exosomes (BxC-e)
  • the induced pluripotent stem cell-derived mesenchymal stem cell (hereinafter, referred to as BxC) culture medium cultured in the above Preparation Example was collected and centrifuged at 300xg for 10 minutes to remove the remaining cells and cell residues.
  • the supernatant was taken and filtered using a 0.22 ⁇ m filter, and then centrifuged at 10,000xg and 4°C for 70 minutes using a high speed centrifuge.
  • the centrifuged supernatant was retaken and centrifuged at 100,000xg for 90 minutes at 4°C using an ultracentrifuge to remove the supernatant.
  • the exosomes remaining in the lower layer were diluted in PBS (phosphate buffered salin) and used in the following experiments.
  • Example 1 For the exosomes isolated in Example 1 (hereinafter, BxC-e), the size distribution of exosomes was confirmed using a nanoparticle tracking assay (NanoSight NS300, Malvern), and the shape of the exosomes was determined using an electron microscope. Confirmed.
  • exosomes derived from BxC of the present invention have properties as exosomes.
  • Human adipocytes (primary human adipocyte, ATCC, USA) were dispensed into a 6-well plate, and 1% penicillin-streptomycin and 10% CS were added in DMEM medium (Gibco, USA) at 37° C., 5% CO 2. It was incubated in the incubator until it grows in a confluent state (up to 6 days).
  • adipose differentiation medium [34 ⁇ M pantothenate (Sigma), 66 ⁇ M biotin (biotin, Sigma) in DMEM medium (Gibco, USA) added with 1% penicillin-streptomycin and 10% FBS , 0.5mM insulin (Sigma), 1mM dexamethasone (dexamethasone, Sigma) and 0.05M IBMX (Sigma)] was added to the culture medium for 5 days.
  • fat differentiation medium [34 ⁇ M pantothenate (Sigma), 66 ⁇ M biotin (biotin, Sigma), 0.5 mM insulin (Sigma) and 1 mM dexamethasone (dexamethasone, Sigma)] was added to DMEM medium (Gibco, USA). It was cultured for an additional 9 days in the medium.
  • negative control is adipocytes cultured for 14 days in DMEM medium supplemented with 10% FBS
  • vehicle control is adipocytes cultured for 14 days in adipogenic conditions without BxC-e treatment
  • BxC-e treatment group is BxC Adipocytes cultured for 14 days in adipose differentiation condition containing -e.
  • the cells were fixed for 1 hour by washing twice with PBS and adding a 10% formalin solution at 400 ul/well. After washing with PBS, an Oil-red O working solution was added per 400 ul/well to stain fat in differentiated adipocytes for 2 hours. Then, after removing the oil-red O working solution and completely removing the oil-red O working solution on the wall of the well with secondary distilled water, put it in a dryer and dry for 5 minutes, and then isopropyl alcohol. Was added to the well so as to be 500 ul/well.
  • Absorbance was measured at 490 nm using a microplate reader (Model 680 microplate reader, Bio-Rad, USA) to compare the amount of fat.
  • exosomes (BxC-e) derived from BxC of the present invention have an excellent effect of inhibiting lipogenesis of adipocytes.
  • Example 2 Isolation of induced pluripotent stem cells-derived mesenchymal stem cells-derived exosomes according to treatment of pretreatment material
  • LANIFIBRANOR 10 ⁇ M-containing culture medium high glucose DMEM (Gibco, Cat no. 11995-065); 10% Fetal bovine Serum (HyClone), 1% MEM Non-Essential Amino Acids Solution (100X) (Gibco, Cat no. 11140-050)] from the induced pluripotent stem cells (iPSC)-derived mesenchyme Stem cells (BxC) were cultured for 24 hours.
  • iPSC induced pluripotent stem cells
  • BxC mesenchyme Stem cells
  • FBS fetal bovine serum
  • the BxC culture medium treated with the pre-treatment material was collected and centrifuged at 300xg for 10 minutes to remove remaining cells and cell residues.
  • the supernatant was taken and filtered using a 0.22 ⁇ m filter, and then centrifuged at 10,000xg and 4°C for 70 minutes using a high speed centrifuge.
  • the centrifuged supernatant was retaken and centrifuged at 100,000xg for 90 minutes at 4°C using an ultracentrifuge to remove the supernatant.
  • the exosomes remaining in the lower layer were diluted in PBS (phosphate buffered salin) and used in the following experiments.
  • Exendin-4 (20nM) was treated instead of LANIFIBRANOR of Example 2-1, and exosomes were isolated in the same manner as in Example 2-1.
  • Example 2 For each exosome (BxC-V37e and BxC-G63e) isolated in Example 2, the size distribution of exosomes was confirmed using a nanoparticle tracking assay (NanoSight NS300, Malvern), and exosomes were used using an electron microscope. I confirmed the shape of the moth.
  • BxC-derived exosomes according to the IVA337 treatment of the present invention (Figs. 3a and 3b) and BxC-derived exosomes (Figs. 4a and 4b) according to Exendin-4 treatment have properties as exosomes Can be seen.
  • Example 1 The exosomes (BxC-e) isolated in Example 1 and the exosomes (BxC-V37e) isolated in Example 2-1 were tested as follows.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • Gibco Gibco (Grand Island, NY, USA)
  • Fatty acid-free bovine serum albumin (BSA), palmitate, and oleate without free fatty acid were purchased from Sigma (St. Louis, MO, USA).
  • HepG2 Human liver cell line
  • HepG2, ATTC Human hepatocyte cell lines
  • DMEM medium containing 10% FBS (Gibco) and 1% penicillin-streptomycin at 37° C. and 5% CO 2 .
  • the cultured cells were dispensed into a 6-well plate at a certain number (5x 10 5 cells/1000 ⁇ l well), and the cells were completely attached to the wells to form a cell shape and then used when the cells reached 95% saturation. .
  • a 1% (w/v) BSA solution containing no free fatty acid prepared in (2) was used as a solvent, and the concentration of the fatty acid prepared in (1) was 100 mM.
  • [1M palmitate 10 ⁇ l + 1% (w/v) BSA 290 ⁇ l (33mM) prepared in (1) above] and [1M oleate 10 ⁇ l + 1% (w/v) BSA 140 ⁇ l prepared in (1) above ( 66mM)] was mixed in a heatblock at 70°C in a volume of 1:1.
  • the 1% (w/v) BSA prepared in (2) was used as a control solution.
  • the 16-hour-treated HepG2 cells were washed once with PBS, and 100 ⁇ g of BxC-e isolated in Example and 100 ⁇ g of BxC-V37e isolated in Example 2 were each treated in 1 ml serum-free medium for 24 hours. Then, each of the following experiments was carried out.
  • Human hepatocytes were digested by adding 1 ml of a trizol solution per well of a 6-well plate, and then 200 ⁇ l of chloroform was added and vortexed, followed by centrifugation at 4° C. and 12,000 rpm for 15 minutes. The supernatant was transferred to a new tube and mixed with 500 ⁇ l of isoprophanol. After 50 times of up & down, the mixture was left on ice for 5 minutes, and then centrifuged at 12,000 rpm and 4° C. for 10 minutes to remove the supernatant. 1 ml of 70% ethanol was added to the remaining precipitate, followed by centrifugation at 12,000 rpm and 4° C. for 5 minutes.
  • the tube containing the RNA precipitate was dried while not visible at room temperature, and the RNA pellet was dissolved using nuclease free water.
  • the concentration of extracted RNA samples at 260 nm and 280 nm wavelength was measured using Nanodrop, and cDNA was synthesized using RT premix.
  • Example 4-1 Using the same method as in Example 4-1, the mRNA expression changes of TNF- ⁇ and MCP1 were confirmed.
  • the primers of the used genes are shown in Table 2 below.
  • Example 4-1 Using the same method as in Example 4-1, the change in mRNA expression of CHOP was confirmed.
  • the primers of the used genes are shown in Table 3 below.
  • Exendin-4 and the exosomes (BxC-G63e) isolated in Example 2-2 were tested as follows.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • Gibco Gibco (Grand Island, NY, USA)
  • Fatty acid-free bovine serum albumin (BSA), palmitate, and oleate without free fatty acid were purchased from Sigma (St. Louis, MO, USA).
  • HepG2 Human liver cell line
  • HepG2, ATTC Human hepatocyte cell lines
  • DMEM medium containing 10% FBS (Gibco) and 1% penicillin-streptomycin at 37° C. and 5% CO 2 .
  • the cultured cells were dispensed into a 6-well plate at a certain number (5x 10 5 cells/1000 ⁇ l well), and the cells were completely attached to the wells to form a cell shape and then used when the cells reached 95% saturation. .
  • a 1% (w/v) BSA solution containing no free fatty acid prepared in (2) was used as a solvent, and the concentration of the fatty acid prepared in (1) was 100 mM.
  • [1M palmitate 10 ⁇ l + 1% (w/v) BSA 290 ⁇ l (33mM) prepared in (1) above] and [1M oleate 10 ⁇ l + 1% (w/v) BSA 140 ⁇ l prepared in (1) above ( 66mM)] was mixed in a 1: 1 volume in a 70°C heatblock.
  • the 1% (w/v) BSA prepared in (2) was used as a control solution.
  • the 16-hour-treated HepG2 cells were washed once with PBS, and 20 nM Exendin-4 100 ⁇ g or 100 ⁇ g of BxC-G63e isolated in Example 2 was treated in 1 ml serum-free medium for 24 hours, and then each of the following experiments was performed. Proceeded.
  • the tube containing the RNA precipitate was dried while not visible at room temperature, and the RNA pellet was dissolved using nuclease free water.
  • the concentration of extracted RNA samples at 260 nm and 280 nm wavelength was measured using Nanodrop, and cDNA was synthesized using RT premix.
  • Example 5-1 Using the same method as in Example 5-1, the mRNA expression changes of TNF- ⁇ and IL-10 were confirmed.
  • the primers of the used genes are shown in Table 5 below.
  • Example 5-1 Using the same method as in Example 5-1, the change in mRNA expression of CHOP was confirmed.
  • the primers of the used genes are shown in Table 3 above.
  • Example 2-1 The exosomes (BxC-V37e) isolated in Example 2-1 and the exosomes (BxC-G63e) isolated in Example 2-2 were tested as follows.
  • mice Six-week-old C57BL/6NHsd male mice were supplied with an MCD diet for 5 days, followed by a normal diet for 2 days. For 12 weeks, the MCD diet and normal food were alternately consumed.
  • test substance was prepared by diluting BxC-G63e or BxC-V37e in PBS.
  • the amount of protein of the test substance from 100 ⁇ g/head to 400 ⁇ g/head was administered intravenously once a day, three times a week, and for 4 weeks.
  • a 6-week-old C57BL/6NHsd male mouse was placed in a calibration frame and fixed, 26 gauge Using a syringe equipped with an injection needle, it was slowly injected at a rate within 1 mL/min through the caudal vein.
  • the liver of the C57BL/6NHsd male mouse administered with the test substance was excised and photographed, and the weight was measured, the right lobe of the liver was fixed in a 10% neutral buffered formalin solution, and the left lobe of the liver was rapidly frozen using liquid nitrogen, It was used in the following experiment.
  • Microvesicular steatosis, macrophobic steatosis, and hypertrophy are observed with a microscope field of 40 magnification or 100 magnification and 0-3 points, respectively, depending on the area occupied by the area to be observed.
  • 5 sites were randomly selected from a 100 magnification field of view, scored 0-3, and compared between groups.
  • microvesicular steatosis Microvesicular steatosis
  • macrophobic steatosis Macrovesicular steatosis
  • all of the NAS scores of hepatocyte hypertrophy hypotrophy
  • the group treated with BxC-G63e or BxC-V37e induces inflammation through the MCD diet, compared to the control group treated with PBS. It was confirmed that it was significantly reduced. In addition, as can be seen in Figure 13a, it was confirmed that the inflammation score was reduced in the group treated with BxC-V37e. Therefore, it was confirmed that the function of inhibiting inflammation of BxC-G63e or BxC-V37e was very excellent.
  • Example 2-1 The exosomes (BxC-V37e) isolated in Example 2-1 and the exosomes (BxC-G63e) isolated in Example 2-2 were tested as follows.
  • TAA at a concentration of 200 mg/kg was administered once a day, three times a week, for 12 weeks.
  • Each test substance was prepared by diluting BxC-G63e or BxC-V37e in PBS.
  • the test substance was administered subcutaneously at 400 ⁇ g/head, once a day, three times a week, for 4 weeks, and at the same time, TAA at a concentration of 200 mg/kg was administered once a day, twice a week, and 4 weeks.
  • TAA at a concentration of 200 mg/kg was administered once a day, twice a week, and 4 weeks.
  • disinfect the administration site with 70% alcohol, pull the skin of the right mouse thigh with the thumb and index finger to create a space between the skin and the muscle, and then place an insulin syringe in the subcutaneous space between the thumb and index finger from the front of the animal. It was stabbed and administered as it was.
  • the liver of C57BL/6 male mice administered with the test substance was removed and photographed, and the weight was measured, the right lobe of the liver was fixed in a 10% neutral buffered formalin solution, and the left lobe of the liver was rapidly frozen using liquid nitrogen, It was used in the following experiment.
  • the fixed tissue was prepared for histopathological examination through general tissue processing procedures such as rectification, dehydration, paraffin embedding, and thinning, and then hematoxylin & eosin (H&E) and picrosirius red staining. was carried out, and histopathological changes were observed using an optical microscope (Olympus BX43, Japan).
  • liver tissue surface of the TAA-PBS group was not smooth compared to the normal group without any treatment, and when treated with BxC-G63e or BxC-V37e, the liver tissue recovered significantly. I was able to observe it.
  • liver tissue damaged by TAA was remarkably recovered by BxC-G63e or BxC-V37e.
  • BxC-G63e or BxC-V37e has very good anti-fibrosis function.
  • the BxC-e, BxC-V37e and BxC-G63e of the present invention inhibit adipogenesis, inhibit inflammation, and inhibit endoplasmic reticulum stress in steatosis-induced hepatocytes. It can be seen that the prevention or treatment efficacy of alcoholic steatohepatitis is excellent.
  • the present invention is the prevention of non-alcoholic steatohepatitis comprising as an active ingredient exosomes derived from mesenchymal stem cells differentiated from induced pluripotent stem cell-derived mesenchymal stem cell progenitor cells with or without pretreatment It relates to a composition for treatment.

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Abstract

La présente invention concerne une composition pharmaceutique pour la prévention ou le traitement de la stéatohépatite non alcoolique, la composition comprenant, en tant que principe actif, des exosomes isolés à partir de cellules souches mésenchymateuses dérivées de cellules souches pluripotentes induites qui ont été ou n'ont pas été traitées avec un matériau de prétraitement. Les exosomes de la présente invention présentent un effet encore amélioré de prévention ou de traitement de la stéatohépatite non alcoolique, par rapport à ceux isolés à partir de cellules souches mésenchymateuses classiques et, en tant que tels, peuvent être avantageusement utilisés pour la recherche et le développement associés et la productisation.
PCT/KR2020/010640 2019-08-22 2020-08-12 Composition comprenant des exosomes dérivés d'un précurseur de cellule souche mésenchymateuse dérivée d'une cellule souche pluripotente induite pour la prévention ou le traitement de la stéatohépatite non alcoolique Ceased WO2021033990A1 (fr)

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US17/621,743 US20220233601A1 (en) 2019-08-22 2020-08-12 Composition comprising exosomes derived from induced pluripotent stem cell-derived mesenchymal stem cell progenitor for prevention or treatment of non-alcoholic steatohepatitis
EP20855301.6A EP4019027B1 (fr) 2019-08-22 2020-08-12 Composition comprenant des exosomes dérivés de cellules souches mésenchymateuses dérivée d'une cellule souche pluripotente induite pour la prévention ou le traitement de la stéatohépatite non alcoolique
CN202080043654.6A CN114025776B (zh) 2019-08-22 2020-08-12 包含源自诱导多能干细胞来源间充质干细胞前体细胞的外泌体的非酒精性脂肪肝炎预防或治疗用组合物
JP2022510957A JP7534055B2 (ja) 2019-08-22 2020-08-12 人工多能性幹細胞由来間葉系幹細胞前駆細胞に由来するエキソソームを含む非アルコール性脂肪性肝炎の予防又は治療用組成物

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EP4190338A4 (fr) * 2020-07-29 2024-03-27 Brexogen Inc. Composition comprenant des exosomes dérivés de cellules souches et sa méthode de production
CN112891543A (zh) * 2021-03-05 2021-06-04 天津医科大学朱宪彝纪念医院(天津医科大学代谢病医院、天津代谢病防治中心) Rassf4作为糖尿病合并非酒精性脂肪性肝病及肝癌治疗的靶点及应用
CN112891543B (zh) * 2021-03-05 2022-05-17 天津医科大学朱宪彝纪念医院(天津医科大学代谢病医院、天津代谢病防治中心) Rassf4作为糖尿病合并非酒精性脂肪性肝病及肝癌治疗的靶点及应用

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